The severe acute respiratory syndrome coronavirus (SARS-CoV) is highly pathogenic in humans, with a death rate near 10%. This high pathogenicity suggests that SARS-CoV has developed mechanisms to overcome the host innate immune response. It has now been determined that SARS-CoV open reading frame (ORF) 3b, ORF 6, and N proteins antagonize interferon, a key component of the innate immune response. All three proteins inhibit the expression of beta interferon (IFN-␤), and further examination revealed that these SARS-CoV proteins inhibit a key protein necessary for the expression of IFN-␤, IRF-3. N protein dramatically inhibited expression from an NF-B-responsive promoter. All three proteins were able to inhibit expression from an interferon-stimulated response element (ISRE) promoter after infection with Sendai virus, while only ORF 3b and ORF 6 proteins were able to inhibit expression from the ISRE promoter after treatment with interferon. This indicates that N protein inhibits only the synthesis of interferon, while ORF 3b and ORF 6 proteins inhibit both interferon synthesis and signaling. ORF 6 protein, but not ORF 3b or N protein, inhibited nuclear translocation but not phosphorylation of STAT1. Thus, it appears that these three interferon antagonists of SARS-CoV inhibit the interferon response by different mechanisms.In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) infected thousands of people throughout the world, killing hundreds. The molecular mechanisms governing virus-induced pathology have not been fully elucidated. The first immune challenge a virus must surmount in order to cause disease symptoms in people is the innate immune response. A major component of innate immunity is the interferon response. Infection of cells with virus causes the activation of several cellular transcription factors, such as IRF-3 and NF-B, which activate the expression of the interferon genes. Once interferon is synthesized and released from cells, it binds to interferon receptors, initiating a signaling cascade of the JAK/ STAT pathway that results in activated transcription factors translocating to the nucleus. These transcription factors bind to and activate genes containing an interferon-stimulated response element (ISRE) in their promoters. Activation of these genes enables the cell to combat the virus infection and can prevent viral replication (8). Many viruses have developed mechanisms to subvert the interferon response. Infection of cells with SARS-CoV does not result in the production of interferon, and pretreatment of cells with interferon prevents growth of 33). These results indicate that SARS-CoV has evolved to overcome the interferon response.SARS-CoV contains a 29.7-kb single-stranded RNA genome wrapped in a helical nucleocapsid composed of multiple copies of N protein, which in turn is surrounded by an envelope containing a 180-to 190-kDa S glycoprotein, a 23-kDa M glycoprotein, an ϳ30-kDa 3a glycoprotein, and a small E protein. The viral gene order is similar to that in other known coronavi...

The host innate immune response is an important deterrent of severe viral infection in humans and animals. Nuclear import factors function as key gatekeepers that regulate the transport of innate immune regulatory cargo to the nucleus of cells to activate the antiviral response. Using severe acute respiratory syndrome coronavirus (SARS-CoV) as a model, we demonstrate that SARS-COV ORF6 protein is localized to the endoplasmic reticulum (ER)/Golgi membrane in infected cells, where it binds to and disrupts nuclear import complex formation by tethering karyopherin alpha 2 and karyopherin beta 1 to the membrane. Retention of import factors at the ER/Golgi membrane leads to a loss of STAT1 transport into the nucleus in response to interferon signaling, thus blocking the expression of STAT1-activated genes that establish an antiviral state. We mapped the region of ORF6, which binds karyopherin alpha 2, to the C terminus of ORF6 and show that mutations in the C terminus no longer bind karyopherin alpha 2 or block the nuclear import of STAT1. We also show that N-terminal deletions of karyopherin alpha 2 that no longer bind to karyopherin beta 1 still retain ORF6 binding activity but no longer block STAT1 nuclear import. Recombinant SARS-CoV lacking ORF6 did not tether karyopherin alpha 2 to the ER/Golgi membrane and allowed the import of the STAT1 complex into the nucleus. We discuss the likely implications of these data on SARS-CoV replication and pathogenesis.

Reovirus infection is initiated by interactions between the attachment protein 1 and cell surface carbohydrate and junctional adhesion molecule A (JAM-A). Expression of a JAM-A mutant lacking a cytoplasmic tail in nonpermissive cells conferred full susceptibility to reovirus infection, suggesting that cell surface molecules other than JAM-A mediate viral internalization following attachment. The presence of integrinbinding sequences in reovirus outer capsid protein 2, which serves as the structural base for 1, suggests that integrins mediate reovirus endocytosis. A ␤1 integrin-specific antibody, but not antibodies specific for other integrin subunits, inhibited reovirus infection of HeLa cells. Expression of a ␤1 integrin cDNA, along with a cDNA encoding JAM-A, in nonpermissive chicken embryo fibroblasts conferred susceptibility to reovirus infection. Infectivity of reovirus was significantly reduced in ␤1-deficient mouse embryonic stem cells in comparison to isogenic cells expressing ␤1. However, reovirus bound equivalently to cells that differed in levels of ␤1 expression, suggesting that ␤1 integrins are involved in a postattachment entry step. Concordantly, uptake of reovirus virions into ␤1-deficient cells was substantially diminished in comparison to viral uptake into ␤1-expressing cells. These data provide evidence that ␤1 integrin facilitates reovirus internalization and suggest that viral entry occurs by interactions of reovirus virions with independent attachment and entry receptors on the cell surface.

It was recently shown that the 7a protein of severe acute respiratory syndrome coronavirus induces biochemical changes associated with apoptosis. In this study, the mechanism by which the 7a protein induces apoptosis was examined. The 7a protein was tested for the ability to inhibit cellular gene expression because several proapoptotic viral proteins with this function have previously been identified. 7a protein inhibited expression of luciferase from an mRNA construct that specifically measures translation, whereas inhibitors of transcription and nucleocytoplasmic transport did not. The inhibition of translation and other cellular processes of gene expression have been associated with the induction of a stress response in cells. Western blot analysis using phosphospecific antibodies indicated that 7a protein activated p38 mitogen-activated protein kinase (MAPK), but not c-Jun N-terminal protein kinase/stress-activated protein kinase. Taken together, these data indicate that the induction of apoptosis by the 7a protein may be related to its ability to inhibit cellular translation and activate p38 MAPK.

There is a substantial need to develop better influenza virus vaccines that can protect populations that are not adequately protected by the currently licensed vaccines. While live attenuated influenza virus vaccines induce superior immune responses compared to inactivated vaccines, the manufacturing process of both types of influenza virus vaccines is time consuming and may not be adequate during a pandemic. Adjuvants would be particularly useful if they could enhance the immune response to live attenuated influenza virus vaccines so that the amount of vaccine needed for a protective dose could be reduced. The glycolipid, alpha-galactosylceramide (alpha-GalCer), has recently been shown to have adjuvant activity for both inactivated and replicating recombinant vaccines. The goal of these experiments was to determine whether a derivative of alpha-GalCer, alpha-C-galactosylceramide (alpha-C-GalCer) can enhance the immune response elicited by a liveattenuated influenza virus vaccine containing an NS1 protein truncation and reduce the amount of vaccine required to provide protection after challenge. Our results indicated that the adjuvant reduced both morbidity and mortality in BALB/c mice after challenge with wild type influenza virus. The adjuvant also increased the amount of influenza virus specific total IgG, IgG1, and IgG2a antibodies as well as IFN-gamma secreting CD8 + T cells. By using knockout mice that are not able to generate NKT cells, we were able to demonstrate that the mechanism of adjuvant activity is dependent on NKT cells. Thus, our data indicate that stimulators of NKT cells represent a new avenue of adjuvants to pursue for live attenuated virus vaccines.